universal primers for PCR ribotyping - (Mar/19/2012 )
We have isolated a bacterium from dung for the biogas production. Biochemical tests indicated that it could be Methanosarcina. I want to perform 16s PCR-Ribotyping. can any one suggest the universal primers.
Hi Thank for response but there is a list of primers which set is best to use and can I use the same set of primers for Archaea and Bacteria ?
Choose one f primer (such as 27f) and another r primer (such as 1492r) and do pcr. Sequence the resulting band with 27f in one reaction and with 1492r in another. Combine the reads, and blast the sequence.
I'd recommend you read some of the references and consult some of the listed web sites, which will explain the universality of these primers, and likely suggest specific ones for Archaea. Or you could look for the Woese paper in which he discovered the Archaea this way.
Thanks veteran. It is helpful
Hi i need to know one thing. Normally in PCR we add both primer in the same reaction tube. Am I supposed to run reaction separately for each primer. Please let me know. Regards
For the PCR, you need both primers present. For sequencing the pcr product, you need only one primer (but probably want to sequence from both ends, so do two reactions, one with each of the primers).